Microsomal Prostaglandin E Synthase1Promotes Hepatocarcinogesis Via EP2Signal Pathway

Objective: Huh-7cells with forced overexpression of mPGES1was constructed bythe lentiviral vector. Overexpression of mPGES1significantly increased the growth,invasion, metastasis in Huh7cell in vitro and in vivo. Also, the levels of PGE2products and EP2expression were detected to elucidate the mechanism of mPGES1involved in hepatocarcinogsis.Method:(1) The full length of mPGES1cloned into the transcripted carrier in order toconstruct recombinant plasmid. The293T cells were co-transfected with lentiviralpackaging helper plasmid and recombinant plasmid. Then the supernatant obtainedand concentrated.(2) Huh7cells were transfected with mPGES1vector (pGC-LV-mPGES1-GFP).Significant overexpression of mPGES1in these stably transfected cells wereconfirmed by RT-PCR and Western blot. The effects of mPGES1overexpression onproliferation, adherence, migration and invasion were detected by MTT and Transwelltechnique.(3) The stably transfected Huh-7cells were injected subcutaneously into BALB/cnude mice. The size of the tumors were observed once every three days until beenstripped in the21th day.(4) The levels of PGE2product and EP receptors expression were detected byELISA, Real-ime PCR and Western blotting.Results:(1) The lentiviral vector(PGC-LV-mPGES1-GFP) been constructed successfully,which biology titer is2×108TU/mL.(2) Compared with the empty vector group and uninfected group, in theoverexpression group mPGES1mRNA and protein expression increased. (3) Compared with the empty vector group and uninfected group, Huh-7cells withmPGES1overexpression showed higher proliferation, adhesion, invasion andmigration abilities in vitro.(4) Huh-7cells with mPGES1overexpression displayed higher proportion of cells inS phase than the empty vector group and uninfected group by flow cytometry.(5) In xenograft model of BALB/c nude mice, mPGES1overexpressed tumors grewfaster then the other two groups. And, the tumor weight increasded approximatelyfour folds(422.2%) compared to the other groups in the21th day.(6) The levels of PGE2products and EP2expression increased in overexpressiongroup compared with the empty vector group and uninfected group, and the level ofEP1,EP3,EP4expression are no significant difference.(7) The levels of PGE2products and EP2expression reduced in mPGES1-shRNAgroup compared with the mPGES1-NC group and control group, and the levels ofEP1, EP3, EP4expression are no significant difference.Conclusion:1. Lentiviral vectors (pGC-LV-mPGES1-GFP) was successfully constructed andefficiently transfected into Huh-7cells, which stably overexpressed mPGES1.2. Forced overexpression of mPGES1in Huh-7cells promoted tumor cellproliferation, adhesion, migration and invasion in vitro.3. In xenograft model, overexpression of mPGES1in Huh-7cells promoted tumor cellgrowth in vivo.4. Mechanistucally, mPGES1might enhance Hepatocarcinogesis through thePGE2-EP2pathway.